4 replies to this topic

[kbpqc]

Hi All,  
We are following the USP Typsin Activity Assay.  Trypsin is suppose to convert the substrate BAEE + H2O to Na-Benzoyl-L-Arginine + Ethanol and the change should be seen via UV Spec at 253nm at a rate of 0.003absorbance units/min.  We are seeing no change at all.  Anyone have any ideas why this change might not be taking place?  We are using a Sigma Trypsin reconsituted in 0.001N HCL.  Thanks.

[Prep!]

does sigma say to reconstitute in HCl??
we used to do it in water.. but not for the trypsin assay but for peptie map and it worked well.. cause i think if i remember right , the optimum pH for its action is around 8. HCl might just not let it be active..
please do cross-check..
Best regards!!!

[kbpqc]

Pradeep Iyer, on Nov 4 2009, 11:26 AM, said:

does sigma say to reconstitute in HCl??
we used to do it in water.. but not for the trypsin assay but for peptie map and it worked well.. cause i think if i remember right , the optimum pH for its action is around 8. HCl might just not let it be active..
please do cross-check..
Best regards!!!

Thanks,  Sigma and USP both say HCL, the first time we tried the assay we used water to reconsitute....still no change.  Sigma thought it might be because it was not in HCL.

[kbpqc]

Problem solved, the cuvettes we were using were not UV trans.  Argh

[Prep!]